Folic acid is essential for normal embryonic development because it participates in one-carbon metabolism for the synthesis of nucleic acids and amino acids required by highly proliferative embryonic cells (Lucock, Mol. Genet. Metab. 71:121 (2000)). Maternal nutrition, especially with regard to folate intake for the prevention of neural tube defects (NTDs), has been the focus of much attention for the past four decades. The early studies linking folate intake to the prevention of NTDs (Hibbard and Smithells, Obstet. Gynaecol. Br. Commonwealth 71:529 (1964); Hibbard and Smithells, Lancet 1, 1254(1965); Smithall et al., Arch. Dis. Child 51:944 (1976)) have been confirmed by recent randomized controlled studies. These studies show that women who were given periconceptional folate supplements had about a 70% reduction in the occurrence and recurrence of NTDs (Laurence et al., Br. Med. J. (Clin. Res. Ed.) 282:1509 (1981); MRC Vitamin Study Research Group, Lancet 338:131(1991); Czeizel and Dudas, N. Engl. J. Med. 327:1832 (1992)).
However, most mothers who give birth to babies having NTDs and/or other birth anomalies, do not exhibit signs of clinical folate deficiency. Therefore, extensive research has been in progress to identify genetic defects that impair cellular metabolism or uptake of folate. The effect of these genetic defects can be corrected by pharmacologic intake of folic acid (Kirke et al., Lancet 348:1037 (1996)). Pharmacologic intake of folic acid raises the plasma concentration of the vitamin and provides sufficient mother-to-fetus transport of folate to bypass impaired folate uptake and/or intracellular metabolism, thereby reducing the occurrence of folate-sensitive congenital abnormalities. Thus far, a number of candidate genes encoding some of the enzymes related to folate metabolic pathways have been identified in association with NTDs. These genes, however, account for only a small number of birth defects (van der Put et al., Exp. Biol. Med. (Maywood), 226:243 (2001)).
Decreased folate uptake by maternal and/or fetal placental cells and embryonic cells in the presence of normal or a low-level blood folate, may be caused by quantitative or functional defects in the membrane proteins required for the uptake of folate. No genetic abnormalities that alter expressions of these membrane proteins have been unambiguously identified (De Marco el al., Am. J. Med. Genet. 95:216 (2000); Barber et al., Am. J. Med. Genet. 76:310 (1998)).
Cellular uptake of folate is mediated via two distinct pathways: the reduced folate carrier (RFC) (Henderson, Annu. Rev. Nutr. 10:319 (1990)), which is an integral transmembrane protein that is present in most cells, and the folate receptor (FR), which is anchored to the plasma membrane of cells by a glycosylphosphatidylinositol (GPI) adduct that internalizes folate by endocytosis of the folate-receptor complex (Antony, Annu. Rev. Nutr. 16:501 (1996)). There are three isoforms of the FR (α, β, γ), which are expressed at different levels in tissues and have different affinities to folate. Cellular uptake of folate depends on the expression levels of each folate receptor isoform (Ross et al., Cancer 73:2432 (1994)).
The contributions of the FRs and the RFC to the cellular uptake of folate during embryogenesis were not appreciated until Piedrahita et al. demonstrated that the ortholog of the human FRα in the mouse (Folbp1) is essential for embryonic organogenesis while the mouse Folbp2, which is the ortholog of the human FRβ, appears to have no function in embryogenesis (Piedrahita, et al., Nat. Genet. 23: 228 (1999)). Nullizygous Folbp1 knockout mouse embryos (Folbp1−/−) had significant congenital malformations and none survived beyond gestation day 10, while nullizygous Folbp2−/−, heterozygous Folbp1+/− or Folbp2+/− embryos, showed no difference in development and viability as compared to wild type embryos (Piedrahita, et al.). Knockout of the RFC gene also proved lethal to embryos (Zhao, et al., J. Biol. Chem. 276:10224 (2001)). Heterozygous RFC dams that were given folic acid produced normal full term nullizygous RFC (−/−) offspring. These studies indicated that both RFC and FR pathways for folate uptake are essential for fetal development.
The FRα is expressed in human placental syncytiotrophoblasts. High concentrations of both FRα and FRβ isoforms are found in maternal placental tissue (Prasad et al., Biochim. Biophys. Acta. 1223:71 (1994)). The essential function of the FRs in human embryogenesis is to ensure cellular uptake of folate. It has been reported that mother-to-fetus transfer of folate is mediated via the FRα (Clark et al., Hum. Reprod. Update 7:501 (2001)). This prompted studies of the FRα gene as a candidate gene responsible for a folate sensitive birth defect, such as a neural tube defect. However, no consistent nucleotide polymorphisms or mutations that affect expression of the FR gene, or the function of the FR protein, have been identified (Barber et al.; De Marco et al.) that could account for the occurrence rate of NTDs. Instead, only a small fraction of women with a NTD-complicated pregnancy were shown to have a polymorphism in the gene encoding folate-dependent enzymes, such as methylene-tetrahydrofolate reductase (MTHFR) (Christensen et al., Am. J. Med. Genet. 84(2):151-57 (1999)).
Since genetic studies have not provided evidence that a mutation of relevant genes (encoding enzymes or FRs) are a significant cause of congenital dysmorphogenesis, it is, therefore, possible that NTDs and other folate-sensitive abnormalities could be autoimmune disorders. Autoantibodies to several proteins have been associated with infertility, miscarriages and fetal abnormalities (Coulam, Early Pregnancy 4:19 (2000); Clark et al., Hum. Reprod. Update 7:501 (2001)). Several previous studies demonstrated that antibodies raised in a rabbit to kidney, heart muscle, testes, placenta and other rat tissues, caused dose-dependent congenital defects and embryonic resorptions when administered to pregnant rats (Brent et al., Proc. Soc. Exp. Bid. Med. 106:523 (1961); Barrow and Taylor, J. Exp. Zool. 176:41 (1971); Brent, Proc. Soc. Exp. Biol. Med. 125:1024 (1967)). The mechanism by which these anomalies occurred was not established. But the administered antibodies were concentrated on the yolk sac, suggesting that the antibodies interfered with delivery of nutrients to the embryo (Slotnick and Brent, J. Immunol. 96:606 (1966)). Thus, it was speculated that antibodies which block the folate binding sites on the FRs (da Costa and Rothenberg, Biochim. Biochim. Acta. 1292:23 (1996)) could interfere with the cellular uptake of folate. Such interference would then impair intracellular folate homeostasis that is essential for normal embryogenesis and fetal development.
There is evidence that women who have had a spontaneous or induced abortion, or a later miscarriage, have an increased risk of a fetal NTD complication in a subsequent pregnancy (Evans, Brit. Med. J. 1: 975, (1979); Carmi et al., Am. J. Med. Genet. 51: 93, (1994); Cuckle, Prenat. Diagn. 3: 287, (1983)).
The cause of NTD(s) is multifactorial and includes chemotherapeutic drugs, especially the antifolates (Hernandez-Diaz et al., N Engl J Med. 343:1608-14, (2000)), anti-epileptic drugs (Dansky et al., Neurology 42: 32-42 (1992)) chromosomal abnormalities (Seller, Clin Dysmorphol. 4:202-07 (1995)), environmental (Finnell et al., Ann NY Acad Sci. 919:261-77 (2000)) and genetic factors (De Marco and Moroni, Am J Med Genet. 95: 216-23 (2000)). Studies that have shown a reduction of about 70% in the occurrence of NTDs with folic acid supplementation beginning at the time of conception (MRC Vitamin Study Research Group, supra) provide evidence that folate circumvents either an impaired intracellular folate-dependent enzymatic pathway, or an inhibitor of the cellular uptake of folate. There is, however, no evidence for diminished function of an enzymatic pathway that could account for a 70% decrease in the occurrence of NTD with the folate supplementation. It is also not known whether the folate-sensitive disorders are due to interference of folate uptake by autoantibodies to the FRs. Therefore, a woman starting a pregnancy does not know whether grain or pharmacologic folate supplements would help to prevent congenital defects, such as a neural tube defect.
The present invention relates to the discovery that folate-sensitive disorders or conditions, such as infertility, spontaneous abortion, unsuccessful in vitro fertilization, or birth defects, are due to interference of folate uptake by an autoantibody against the folate receptor. The present invention provides a reliable assay to detect autoantibodies to folate receptors in a mammal, especially in a human.